Autoregulation of the Raf-1 serine/threonine kinase

Cutler, Richard E.; Stephens, Robert M.; Saracino, Misty; Morrison, Deborah K.

doi:10.1073/pnas.95.16.9214

Cited by 174 publications

(153 citation statements)

References 49 publications

Supporting

Mentioning

143

Contrasting

Unclassified

Order By: Relevance

“…Our finding that the S365A mutation increases not only the biological activity of B-Raf wt but also rescues that of B-Raf with a neutralized N-region (Figure 3b and c) strongly supports a model in which a major function of the N-region is to oppose the transition into an inactive conformation stabilized by binding of a 14-3-3 dimer to S365 and S729. Our model (Figure 9a), similar to that suggested by Mercer and Pritchard (2003), is consistent with recent studies showing that the negatively charged N-region of Raf-1 relieves autoinhibition by the N-terminal moiety owing to its reduced affinity towards a CR3 with a charged N-region (Cutler et al, 1998;Tran and Frost, 2003). However, we accept that similar experiments led to the alternative conclusion that the charged N-region induces a conformational change that does not affect the interaction between N-and C-terminal moiety but renders the CR3 resistant to the autoinhibition imposed by the N-terminal moiety (Chong and Guan, 2003).…”

Section: Regulation Of B-raf Signallingsupporting

confidence: 92%

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

et al. 2006

View full text Add to dashboard Cite

The BRAF V600E mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf wt ), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5 0 -triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf V600E , although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf V600E was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf wt and B-Raf V600E and suggests that B-Raf V600E cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation. Keywords: Ras; 14-3-3 proteins; b-Catenin; E-cadherin; mammary epithelial cells IntroductionThe Ras/Raf/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal regulated kinase (ERK) pathway plays a pivotal role in control of proliferation and differentiation and, owing to its role as a gatekeeper of this pathway, Raf appears an attractive therapeutic target (O'Neill and Kolch, 2004;Wilhelm et al., 2004). The Raf-kinase family comprises the A-Raf, B-Raf and Raf-1 isoforms in vertebrates as well as D-Raf and LIN-45 in Drosophila and Caenorhabditis, respectively. B-Raf, the major ERK activator in vertebrates, is required for the maintenance of basal ERK activity and displays the most potent transforming activity (Papin et al., 1998;Brummer et al., 2002;Mercer and Pritchard, 2003). All isoforms share three highly conserved regions (CRs; Figure 1a): the N-terminal CR1 contains the Ras-guanine 5 0 -triphosphate (GTP)-binding domain (RBD), which initiates the interaction with Ras-GTP through a conserved arginine residue (R188 in B-Raf) that is required for the recruitment and activation of Raf at the plasma membrane. Consequently, mutation of this residue prevents Ras/Raf interaction and renders D-Raf, B-Raf and Raf-1 unresponsive to most extracellular signals (Hou et al., 1995;Marais et al., 1997;Brummer et al., 2002). The ...

show abstract

Section: Regulation Of B-raf Signallingsupporting

confidence: 92%

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

et al. 2006

View full text Add to dashboard Cite

show abstract

“…D279 and its context do not fit the proposed consensus motif for a caspase-9 cleavage site (Thornberry et al, 1997;Garcia-Calvo et al, 1999), but the autocatalytic cleavage site (PEPDA) in caspase-9 itself also exhibits poor similarity with the consensus sequence (Stennicke et al, 1999). Previous work has shown that Raf-1 contains an autoinhibitory domain within the first 330 amino acids of its N-terminus (Cutler et al, 1998;. Deletions of the N-terminal regulatory domain have been found in several activated forms of Raf genes detected in certain neoplastic human cells (Stanton and Cooper, 1987), resulting in a constitutively active kinase domain that is comparable to the oncogenic v-Raf protein (Cleveland et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

Apoptosis of hematopoietic cells induced by growth factor withdrawal is associated with caspase-9 mediated cleavage of Raf-1

et al. 2005

View full text Add to dashboard Cite

The Raf-1 serine/threonine kinase is a key protein that is implicated in the transmission of many growth and cell survival signals. In the present study we demonstrate that apoptosis of hematopoietic cells induced by IL-3-deprivation is associated with the cleavage of Raf-1, resulting in the separation of the N-terminal regulatory domain and the C-terminal kinase domain. Raf-1 cleavage specifically occurs upon triggering of the mitochondrial death pathway, and coincides with the activation of specific caspases. Moreover, Bcl-2 overexpression or treatment with the caspase inhibitor z-VAD.fmk completely prevented Raf-1 cleavage, whereas caspase inhibition by treatment of cells with Ac-DEVD.fmk or z-IETD.fmk, or CrmA overexpression had no effect. Furthermore, in vitro cleavage studies indicate that caspase-9, which is the apical protease in the mitochondrial death pathway, is able to cleave Raf-1 at position D279. Cell fractionation studies showed that the Raf-1 C-terminal fragment that is generated upon IL-3 withdrawal is localized predominantly to the mitochondria. In addition, constitutive expression of this Cterminal Raf-1 fragment fused to a mitochondrial targeting sequence in Ba/F3 pre-B cells significantly delays apoptosis induced by IL-3 withdrawal. These results suggest an important role for caspase-9 mediated cleavage of Raf-1 in the negative feedback regulation of hematopoietic cell apoptosis induced by growth factor withdrawal.

show abstract

“…A Ras dimer would elegantly resolve the steric dilemma of how the tiny Ras effector domain can engage two different domains in Raf at the same time. In any case, Ras binding seems to relieve the inhibition which the N-terminal regulatory domain of Raf-1 exerts over the catalytic domain at the C-terminus [34]. Point mutations in the CRD, which can alleviate this inhibition, were isolated in a screen for Raf-1 mutations that increased the affinity for Ras [25].…”

Section: A Complicated Relationship : How Ras Proteins Regulate Raf Kmentioning

confidence: 99%

Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions

Kölch

2000

Biochemical Journal

1,057

118

View full text Add to dashboard Cite

The Ras\Raf\MEK (mitogen-activated protein kinase\ERK kinase)\ERK (extracellular-signal-regulated kinase) pathway is at the heart of signalling networks that govern proliferation, differentiation and cell survival. Although the basic regulatory steps have been elucidated, many features of this pathway are only beginning to emerge. This review focuses on the role of protein-protein interactions in the regulation of this pathway, INTRODUCTIONThe mitogen-activated protein kinase (MAPK) pathway is one of the primordial signalling systems that Nature has used in several permutations to accomplish an amazing variety of tasks. It exists in all eukaryotes, and controls such fundamental cellular processes as proliferation, differentiation, survival and apoptosis. The basic arrangement includes a G-protein working upstream of a core module consisting of three kinases : a MAPK kinase kinase (MAPKKK) that phosphorylates and activates a MAPK kinase (MAPKK), which in turn activates MAPK ( Figure 1). This set-up provides not only for signal amplification, but, maybe even more importantly, for additional regulatory interfaces that allow the kinetics, duration and amplitude of the activity to be precisely tuned. At present we can distinguish six MAPK modules, which share structurally related components, but seem to mediate specific biological responses. For recent reviews on MAPK pathways, the reader is referred to references [1][2][3]. Here we will focus on the regulation of the ERK (extracellular-signal-regulated kinase) pathway, which features Ras as G-protein, Raf as MAPKKK, MEK (MAPK\ERK kinase) as MAPKK and ERK as MAPK.Despite enjoying a decade in the limelight of scientific interest and revealing a plethora of new insights into the circuitry of signalling pathways in general, this pathway still holds many secrets. These pertain mainly to the regulation of Raf, and to a deeper understanding of how specific biological responses are encoded by spatial and temporal changes in the activity and subcellular distribution of the pathway components, and how these fluctuations are orchestrated at the molecular level. Work from many laboratories has highlighted protein-protein interactions as powerful means of co-ordinating signalling processes, most strikingly exemplified by the assembly of multi-protein signalling complexes on activated receptors, or of transcriptionfactor complexes on gene promoters. However, it is becoming Abbreviations used : Bcr, Breakpoint cluster region ; BXB, isolated Raf-1 kinase domain ; CNK, connector-enhancer of KSR ; CK2, casein kinase 2 ; CRD, cysteine-rich domain ; DEF, docking site for ERK, FxFP ; DLK, dual leucine-zipper-bearing kinase ; ERK, extracellular-signal-regulated kinase ; Hsp, heat-shock protein ; KIM, kinase interaction motif ; IκB, inhibitor of NF-κB ; JNK, c-Jun N-terminal kinase ; KSR, kinase suppressor of Ras ; MAPK, mitogen-activated protein kinase ; MAPKK, MAPK kinase ; MAPKKK, MAPK kinase kinase ; MEK, MAPK/ERK kinase ; MEKK, MEK kinase ; MP1, MEK partner 1 ; MUK, MAPK upstream kin...

show abstract

Autoregulation of the Raf-1 serine/threonine kinase

Cited by 174 publications

References 49 publications

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

Apoptosis of hematopoietic cells induced by growth factor withdrawal is associated with caspase-9 mediated cleavage of Raf-1

Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions

Contact Info

Product

Resources

About